Nowadays molecular chaperones play an important role in a wide range of biotechnological applications (Mogk et al. 2002 Chembiochem 3, 807). There are a lot of folding helpers which possess chaperone as well as enzymatic properties. For these reasons they are useful for a variety of practical applications in the field of protein folding.
Chaperones, which are known as classical “folding helpers”, are polypeptides that assist the folding and maintenance of structural integrity of other proteins. They possess the ability to promote the folding of a polypeptide both in vivo and in vitro. Generally, folding helpers are subdivided into folding catalysts and chaperones. Folding catalysts accelerate the rate limiting steps in protein folding due to their catalytic function. Examples of catalysts are further described below. Chaperones are known to bind to denatured, partially denatured, or hydrophobic surfaces of polypeptides and thus help to re-nature proteins or to keep them in solution. Thus, unlike folding catalysts, chaperones exert a mere binding function (Buchner, J., Faseb J 10 (1996) 10-19). Chaperones are ubiquitous stress-induced proteins involved in protein maturation, folding, translocation, and degradation (Gething, M. J. and Sambrook, J., Nature 355 (1992) 33-45). Although also present under normal growth conditions, they are abundantly induced under stress conditions. This further supports the idea that their physiological function is to cope with stress conditions.
To date, several different families of chaperones are known. All these chaperones are characterized by their ability to bind unfolded or partially unfolded proteins and have a physiological function that is linked to the correct folding of proteins or the removal of denatured or aggregated protein.
Well-characterized examples of chaperones are members of the so-called heat-shock families of proteins, which are designated according to their relative molecular weight, for example, hsp100, hsp90, hsp70, and hsp60, as well as the so-called shsps (small heat-shock-proteins) as described by Buchner, J., Faseb J 10 (1996) 10-19, and by Beissinger, M. and Buchner, J., Biol. Chem. 379 (1998) 245-59.
Folding catalysts, unlike chaperones, assist folding by accelerating defined rate-limiting steps, thereby reducing the concentration of aggregation-prone folding intermediates. One class of catalysts, the protein disulfide isomerases (alternatively designated as thiol-disulfide-oxido-reductases), catalyzes the formation or the rearrangement of disulfide bonds in secretory proteins. In gram-negative bacteria, the oxidative folding of secretory proteins in the periplasm is adjusted by a cascade of protein disulfide isomerases designated DsbA, DsbB, DsbC, and DsbD (Bardwell, J. C., Mol Microbiol 14 (1994) 199-205 and Missiakas, D., et al., Embo J 14 (1995) 3415-24).
Another important class of folding catalysts referred to as peptidyl prolyl cis/trans isomerases (PPIs) comprise different members such as CypA, PpiD (Dartigalongue, C. and Raina, S., Embo J 17 (1998) 3968-80, FkpA (Danese, P. N., et al., Genes Dev 9 (1995) 387-98), trigger factor (Crooke, E., and Wickner, W., Proc Natl Acad Sci USA 84 (1987) 5216-20 and Stoller, G., et al., Embo J 14 (1995) 4939-48), and SlyD (Hottenrott, S., et al., J Biol Chem 272 (1997) 15697-701).
Due to sequence similarity and protein topology, prolyl isomerases are divided into three distinct families, the cyclophilins, the FK506 binding proteins (FKBPs), and the parvulins. Cyclophilins bind to and are inhibited by the immunosuppressant cyclosporin A. Parvulins are a family of prolyl isomerases, which are inhibited neither by cyclosporin A nor by FK506. FKBPs bind to and are inhibited by FK506 and rapamycin. (The acronym FKBP stands for “FK506-binding protein.” FK506 is a macrolide that is used as immunosuppressant drug). The first x-ray structure of an FKBP to be determined at high resolution was that of human FKBP12. It is a five-stranded antiparallel β-sheet wrapping with a right-handed twist around a short α-helix. The five-stranded β-sheet framework includes residues 2 to 8, 21 to 30, 35 to 38 with 46 to 49, 71 to 76, and 97 to 106 (van Duyne et al., Science (1991) 252, 839-842). Subsequent research has shown that FKBPs, as well as cyclophilins and parvulins, form a highly conserved family of enzymes found in a wide variety of procaryotic and eucaryotic organisms (for review see John E. Kay, Biochem. J. (1996) 314, 361-385). For instance, 10 prolyl isomerases have been identified in E. coli so far (2 parvulins, 3 cyclophilins and 5 FKBPs).
Usually, FKBPs are defined according to the binding criterion, i.e., they recognize and bind FK506 with high affinity in the nanomolar range. There are, however, FKBP-like domains, which are no more susceptible to prolyl isomerase inhibition by FK506. These FKBP-like domains share significant sequence similarity with FKBP12, but some of the amino acid residues mediating FK506 binding are mutated, and the affinity is shifted to the micromolar range. For instance, SlyD and trigger factor (two cytosolic PPIases from the E. coli cytosol) may be envisaged as FKBP-like proteins. Both prolyl isomerases harbor domains sharing significant sequence homology with FKBP12, but their binding affinity to FK506 is rather poor and lies in the micromolar range (Scholz et al. Biochemistry (2006) 45, 20-33). In terms of sequence similarity and protein topology, however, both SlyD and trigger factor are undoubtedly members of the FKBP family (Wülfing et al., J. Biol. Chem (1994) 269(4) 2895-2901, Callebaut & Mornon. FEBS Lett. (1995) 374(2), 211-215).
FKBP domains and FKBP-like domains may form part of larger molecules with complex topologies. In mammalian cells, FKBP12, FKBP12A, and FKBP13 contain only the basic FKBP domain, while FKBP25 and FKBP52 have one or more FKBP domains as part of a larger molecule (for review see John E. Kay, Biochem. J. (1996) 314, 361-385).
Modularly constructed FKBPs are also found in procaryotic cells, for example, the aforementioned trigger factor consists of three well-separated domains with distinct functions. The N-domain mediates binding to the 50S subunit of the E. coli ribosome (Hesterkamp et al., J Biol Chem. (1997) Aug. 29; 272(35):21865-71). The M (middle) domain harbors the prolyl isomerase active site (Stoller et al., FEBS Lett. 1996 Apr. 15; 384(2):117-22), and the C domain encompasses the polypeptide binding site which mediates binding of extended polypeptide substrates (Merz et al., J Biol Chem. 2006 Oct. 20; 281 (42) 31963-31971). Another example of a modularly constructed peptidyl-prolyl isomerase is the periplasmic FkpA, which consists of an N-terminal chaperone and dimerization domain and a C-terminal FKBP domain (Saul et al., J. Mol. Biol (2004) 335, 595-608).
Some folding helpers comprise both a catalytically active domain as well as a chaperone (or polypeptide binding) domain. For example, the prolyl-isomerases trigger factor (Scholz et al. 1997, Embo J. 16, 54-58; Zarnt et al. 1997, JMB 271, 827-837), FkpA (Saul et al. 2004. JMB 335, 595-608), and SlyD belong to these folding helpers. Recently it could be shown that FkpA and SlyD are remarkably suitable as fusion modules for the production of recombinant proteins. Both chaperones increase the expression rate of their client proteins, support correct refolding and increase the solubility of aggregation-prone proteins like retroviral surface proteins (Scholz et al. 2005, JMB 345, 1229-1241 and WO 03/000877).
FkpA, SlyD, and SlpA are bacterial chaperones that belong to the family of FK506 binding proteins. As mentioned above, FK506 is a macrolide that is used as immunosuppressant drug. The cellular receptors for FK506 are still in the focus of world wide research groups. At the beginning of the 1990s, the three-dimensional structure of a human FKBP, i.e., FKBP12, could be resolved (van Duyne et al. 1991, Science 252, 839-842). In contrast to FkpA, SlyD, and SlpA, the human FKPB12 does not have any chaperone activity, and it only has a modest prolyl isomerase activity.
In many diagnostic applications recombinantly produced proteins are used, e.g., as antigens. These antigens may be produced as fusion proteins containing one part that makes up the antigenic portion or target polypeptide which has to be recognized by a specific binding partner that is present in the sample or in the assay mixture. The oilier part of the recombinantly produced fusion protein is a polypeptide portion that is fused to the antigenic part in order to facilitate the cloning, expression, folding, solubilization or purification of the specific antigen. The synthesis of recombinantly produced fusion proteins is well described in prior art. It is quite common to use chaperones as that part of the fusion protein that functions as a helping molecule for the expression, folding, purification, and solubilization of the target polypeptide. For example, U.S. Pat. No. 6,207,420 discloses a fusion protein system for the expression of heterologous proteins, i.e., the amino acid sequences of the target polypeptides part and the fused peptide part originate from different organisms. WO 03/000878 describes the use of FKBP chaperones as tools for the expression of retroviral surface glycoproteins.
While common methods for the expression, purification, folding and solubilization of fusion proteins seem to work reliably, in particular those methods in which folding helpers are used, there still remain some problems to be solved. For instance, whenever a fusion protein containing non-human amino acid sequences is used as a binding partner in a human diagnostic test, there is still a problem of interferences due to these non-human proteins used. Quite often antibodies that abundantly occur in human blood samples react with bacterial proteins present in the assay reagents. Such interferences may result in high background noise or may even cause wrong test results. Another common problem consists in adapting or optimizing the affinity of the fusion partner to the respective client protein. The affinity of any fusion module for the target part must be well balanced. If the affinity is too high, the fusion protein will be perfectly soluble, but the complex between fusion module and client protein will remain in a closed conformation and will thus be inactive in an immunological assay. If the affinity is too low, the client protein should be accessible and active in an immunoassay, but it will not be sufficiently protected against aggregation.
It was therefore an object of the present invention to provide an expression system that is suitable for producing chaperone-like proteins that may be used in a wide range of biotechnological and in particular in diagnostic and pharmaceutical applications which cause no or only little interferences with molecules and substances present in isolated human samples. The prior art does not disclose an effective folding helper, i.e., a helper that exerts both high catalytic and chaperone activities, that consists mainly of human amino acid sequences.
Although several protein sequence alignments of human and bacterial chaperones exist (Wülfing et al 1994, JBC 269, 2895-2901; Hottenrott et al. 1997, JBC 272, 15697-15701; Suzuki et al. 2003. JMB 328, 1149-1160), if has not yet been shown how an efficient humanized folding helper having dual function, i.e., catalytic and chaperone-like functions, may be generated.